Stabilization of carboxyl esterase

ABSTRACT

Carboxyl esterase is inactivated by several chemical compounds such as naproxen or diclofop. By substituting or modifying certain basic residues of the carboxyl esterase, this enzyme shows improved stability properties during application. In this way it is possible to perform stereospecific hydrolysis reactions on industrial scale even at high substrate concentrations.

This application is a continuation of application Ser. No. 07/884,465filed May 14, 1992, now U.S. Pat. No. 5,238,831, which is a continuationof Ser. No. 07/515,736, filed Apr. 26, 1990, now abandoned, which is acontinuation-in-part of Ser. No. 07/366,124, filed Jun. 14, 1989, nowabandoned.

BACKGROUND AND RELEVANT LITERATURE

U.S. Pat. No. 4,886,750 discloses the use of esterases in thestereoselective hydrolysis of esters of 2-arylpropionic acids. In thisdocument the enzyme responsible for the hydrolysis of (S)-naproxenesters is characterized. The corresponding esterase gene was obtainedfrom the Bacillus subtilis Thai 1-8 strain (CBS 679.85). This geneencoding the enzyme responsible for the stereoselective conversion of(R,S)-naproxen ester was cloned in E. coli and Bacillus subtilis. It wasfound that the esterase activity was improved by introducing multiplegene copies in several Bacillus subtilis (a.o. CBS 673.86). Thesuitability of the microorganism and the enzyme derived therefrom foruse in a process to hydrolyse S-naproxen ester was therefore alsoimproved.

In said U.S. patent only low substrate concentrations (naproxen oribuprofen) are used. In contrast, commercial applications require highproduct concentrations in order to obtain economically attractiveresults. However, during tests at high substrate concentrations(commercial conditions) irreversible inactivation of the enzyme has beennoticed. For example, carboxyl esterase obtained from Bacillus Thai I-8was almost completely inactivated within one hour when 30 g/l naproxenester was added (pH=9, T=40° C. and Tween 80 (TM) medium). The esteraseas such is stable at pH=9 and T=40° C. (with and without Tween 80 (TM))for several hours. During the stereoselective hydrolysis of(R,S)-naproxen ester, the enzyme was inactivated by the naproxen formedduring the hydrolysis. High yields of naproxen could not therefore beobtained.

The enzyme carboxyl esterase may be used in several other stereospecificesterase hydrolysis reactions. However, it is found that the product(the acid) of these reactions often inactivates the enzyme when thereaction takes place at commercially interesting starting concentrationsof the ester.

The carboxyl esterase can be used in the stereospecific hydrolysis ofdiclofop esters, resulting in the corresponding enantiomeric pure(S)-acid, which process is described in EP-A-0299559. The diclofopformed will inactivate the enzyme under commercially attractiveconversion conditions.

Other compounds that inactivate the enzyme are, for example, 2-naphthoxyacetic acid, ibuprofen, 2-naphthol and phenol.

In the literature enzymes are known to become inactivated because oftheir low thermal stability. At elevated temperatures unfolding of theenzyme may take place. Heat treatment causes especially the hydrogenbonds to break (see e.g. R. D. Schmid, Advances in BiochemicalEngineering 12, Ghose, Fiechler & Blakebrough (Eds), Springer, Berlin(1979) pp. 41-115). Thermo unfolding of enzymes can, however, bediminished by immobilization or cross-linking of the enzyme. Forexample, cross-linking with glutaraldehyde improved the thermostabilityof Papain (Royer et al., FEMS Lett. 80 (1977) 1) and Subtilopeptidase(Boudrant et al., Biotechnol. Bioeng. 18 (1976) 1719).

Even the mechanism of thermostabilisation is not well understood. E. T.Reese and M. Manders (Biotechnol. Bioeng. 22 (2) 1980 pp. 326-336 showedthat cross-linking (glutaraldehyde treatment) did not result in anincrease of thermostability and activity of cellulase. Similar resultswere found by N. W. Ugarova (Biokhimiya 42 (7), 1977 pp. 1212-1220) whoreported that modification of peroxidase with glutaraldehyde gave a2.5-fold decrease in thermostability.

The prior art presents only very specific solutions for specificproblems (immobilization and cross-linking techniques) which are notgenerally applicable. Moreover it has been noticed that the carboxylesterase is not thermally inactivated at normal reaction conditions (upto 45° C.) but is only inactivated by certain compounds at reactionconditions. The prior art is silent on such kind of inactivations.

When the amino acid residue which is the cause of inactivation of theprotein is known, an alternative approach to chemical modification isavailable. In that case one can replace the residue for another one bysite-directed mutagenesis, as described for instance by Ausubel et al.(Current Protocols in Molecular Biology, John Wiley & Son Inc., 1987,New York). In this way e.g. the oxidation resistance of B. alcalophilusserine protease was improved by replacing a methionine residue by aserine residue (European patent application 0328229).

The known stabilization techniques cannot be applied as such to thepresent enzyme because the nature of the inactivation is different wheninactivation by chemical compounds plays a role.

SUMMARY OF THE INVENTION

The present invention relates to a modified carboxyl esterase whichshows enhanced stability in the presence of compounds such as naproxen,and may be employed in the stereospecific hydrolysis of such compounds.Accordingly, the present invention provides a process forstereospecifically hydrolysing an optically active substrate whichcomprises hydrolysing the substrate in the presence of a carboxylesterase which shows enhanced stability when contacted with 15 mg/ml of(S)-naproxen at 40° C. for 1.5 hours, compared to the wild type. Thismodified carboxyl esterase can be obtained by substituting or modifyingat least one basic amino acid residue of the wild-type carboxylesterase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence of the coding region of the genefor carboxyl esterase from Bacillus subtilis Thai I-8 (CBS 679.85).

FIG. 2 shows the activity of the wild-type carboxyl esterase and severalmutant carboxyl esterases;

--wild-type carboxyl esterase

+--34 Glu mutant

* --81 Glu mutant

--Lys 217 Glu mutant

FIG. 3 shows the influence of formaldehyde treatment on the activity ofthe carboxyl esterase;

--after formaldehyde treatment

+--after formaldehyde and naproxen treatment.

FIG. 4 shows the conversion of anproxen ester using carboxyl esteraseand the modified carboxyl esterase;

--unmodified enzyme

+--glutaric anhydride modified

*--succinic anhydride modified

□--glyoxal modified

×--glutaric aldehyde modified

⋄--formaldehyde modified.

FIG. 5 shows the conversion of (R,S)-diclofop ethyl ester using carboxylesterase and modified carboxyl esterase, respectively.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

It is to be understood that the term `carboxyl esterase` as usedthroughout the present application denotes an esterase obtainable from aBacillus strain and capable of stereospecifically hydrolysingS-naproxen.

Carboxyl esterase is stable at temperatures up to 45° C. In the presenceof compounds like naproxen the enzyme is quickly inactivated. The enzymesubstantially loses activity within 1.5 hours in the presence of 15mg/ml (S)-naproxen at 40° C. The inactivation of the enzyme isaccompanied by aggregation of the enzyme. This inactivation, ordestabilisation is not due to thermo-inactivation but is found to berelated to the chemical effect of compounds like naproxen on the enzyme.The present invention is based on the discovery that positively chargedamino acid residues in the carboxyl esterase are involved in thedestabilization. Possibly the naproxen acid reacts with the free aminogroups at the surface of the enzyme, thereby allowing the hydrophobicbulk of the naproxen acid to interfere with the folding of the enzyme.This unfolding is noticed as increased susceptibility of the enzyme toproteolytic breakdown in the presence of naproxen. By substituting(protein engineering) or by chemically modifying these basic residues,the positive charge of these amino acids can be removed or reversed.This would prevent the binding of the naproxen acid to the enzyme. Inthis respect it is to be noticed that only small and not too hydrophilicchemical groups can be used to modify the enzyme. Benzaldehyde, forinstance, does not have a positive effect on enzyme stability. Also,changing the positive charged residues to other residues with positivecharge but less susceptible to chemical modification, such as lysine toarginine substitutions (see e.g. R. D. Schmid, Advances in BiochemicalEngineering 12, Ghose, Fiechler & Blakebrough (Eds.), Springer, Berlin(1979), 41-115), might give rise to stabilization of the enzyme.

Accordingly, in a further aspect, the present invention provides amodified carboxyl esterase which has been produced by treating wild-typecarboxyl esterase with a compound comprising at least one group whichcan react with a positively charged basic amino acid residue in thecarboxyl esterase. This results in higher product concentrations andhigher yields. Aldehydes (mono- or dialdehydes) such as formaldehyde,glutaraldehyde or glyoxal, and anhydrides such as glutaric acidanhydride or succinic anhydride, are examples of compounds which may beused to treat the carboxyl esterase.

Generally 0.05-10 v/v% (calculated on the reaction mixture) of thecompound (stabilizing agent) is added to the reaction mixture containingthe wild-type carboxyl esterase. Typically 0.1-5 v/v% of this agent isadded. The pH is maintained during the stabilization of the enzyme at atleast pH=7, typically at a pH from 7 to 10.

It is found that substantially all the carboxyl esterase is stabilizedafter addition of the compound such as aldehyde or anhydride. The factthat stabilization can occur using formaldehyde, a mono aldehyde, or ananhydride, indicates that the enzyme is being chemically modified by theformaldehyde or anhydride rather than intra molecular cross-linked.

According to a still further aspect of the present invention new enzymesare provided, in particular modified carboxyl esterases, which can beobtained by expression of genes encoding said enzyme, which differs fromsaid wild-type esterase in at least one basic amino acid residue presentin the corresponding wild-type enzyme and which exhibit improvedproperties during application. It has surprisingly been found thatcertain lysine, argine and histidine residues are involved in theinactivation of the carboxyl esterase.

The present invention thus provides a stabilized or modified enzyme,particularly a stabilised or modified carboxyl esterase, which has beenproduced by replacing at least one basic amino acid residue in thecorresponding wild-type enzyme, and expressing the mutant gene or whichbasic amino acids are modified by the action of certain chemicalcompounds.

After determining the DNA sequence of carboxyl esterase (see Example 1),lysine, arginine and histidine residues of the esterase can be replacedby mutating the esterase gene with the technique of site directedmutagenesis (Ausubel et al., 1987, Current Protocols in MolecularBiology, John Wiley & Son Inc., New York). In this way the positivelycharged basic lysine and/or arginine residues can, for instance, besubstituted by neutral (non charged) or negatively charged residues(e.g. glutamine, serine or glutamic acid). In the same way otherresidues (e.g. histidine), which are involved in the destabilisation ofcarboxyl esterase, can be substituted by other residues.

The modified enzyme shows improved properties during industrialapplication, for example, in the hydrolysis of naproxen ester. Byimproved properties as used herein we mean a high conversion performancearising from improved stability, and especially improved stabilityagainst certain chemical compounds, relative to the correspondingwild-type enzyme.

"Carboxyl esterase" as used herein means an esterase obtainable from aBacillus strain, which is capable of stereospecifically hydrolysingS-naproxen ester. Preferably, the enzyme is substantially identical, oridentical, to the esterase obtainable from a Bacillus subtilis strain,more preferably from the Bacillus subtilis Thani 1-8 strain (CBS679.85). By an enzyme which is substantially identical to the esteraseobtainable from the Bacillus subtilis Thai I-8 strain is meant that theDNA sequence encoding an esterase has at least 70% homology in thenucleotide sequence with the DNA sequence encoding for the esterase fromBacillus subtilis Thai I-8 strain.

The following equation, which has been derived from analysing theinfluence of different factors on hybrid stability:

    Tm=81+16.6 (log 10 Ci)+0.4 (% G+C)-600/n-1.5 (% mismatch) (Current protocols in molecular biology 1987-1988, edited by Ausubel et al.).

n=length of the shortest chain of the probe

Ci=ionic strength (M)

G+C=base composition

Tm=hybridization temperature

was used to determine the homology which could be detected in ourexperiments. Assuming a probe length of 300 bases, we were able todetect a homologous gene which shows at least 67% homology within afragment of 300 bases or more. In the determination of homologypercentage we assumed that the GC contents of Bacillus is 50% (Normore,1973, in Laskin and Lechevalier (ed), Handbook of Microbiology vol. II,CRC Press, Inc. Boca Raton. Fla.).

This means that a modified carboxyl esterase with at least 70% homologywith Bacillus subtilis Thai I-8 carboxyl esterase is comprised in theinvention.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritand scope of the appended claims.

The following examples further illustrate the invention.

EXAMPLE 1

Determination of the amino acid sequence of carboxyl esterase ofBacillus subtilis 1-85/pNAPT-7 (CBS 673.86)

The amino acid sequence of carboxyl esterase originating from Bacillussubtilis 1-85/pNAPT-7 (CBS 673.86), described in U.S. Pat. No. 4,886,750was determined as follows. The nucleotide sequence of the 2.2HindIII-HindIII insert fragment of pNAPT-7 was determined by the dideoxychain termination method as described by Sanger et al. (Proc. Natl.Acad. Sci. USA 75 (1977), 5463). Within the sequence only one large openreading frame capable of encoding a 30 kD protein could be detected.From the nucleotide sequence of this open reading frame the amino acidsequence of this carboxyl esterase has been derived. FIG. 1 shows theDNA sequence and the derived amino acid sequence for this carboxylesterase. The one letter code for amino acids is explained in thefollowing table:

    ______________________________________                                        A = Alanine        L = Leucine                                                R = Arginine       K = Lysine                                                 N = Asparagine     M = Methionine                                             D = Aspartic acid  F = Phenylalanine                                          C = Cysteine       P = Proline                                                Q = Glutamine      S = Serine                                                 E = Glutamic acid  T = Threonine                                              G = Glycine        W = Tryptophan                                             H = Histidine      Y = Tyrosine                                               I = Isoleucine     V = Valine                                                 ______________________________________                                    

EXAMPLE 2 Mutation of lysine residues in carboxyl esterase.

The DNA fragment encoding carboxyl esterase in Bacillus subtilis ThaiI-8 (CBS 679.85) (BclI-HindIII fragment of 2.0 kb originating frompNAPT-2, see EP-A-233656) was cloned into vector pTZ18R. Single strandedDNA was prepared according to the instructions of supplier (Pharmacia).This single stranded DNA was subjected to oligonucleotide directedmutagenesis as described (Ausubel et al., ibid.). Eleven differentmutagenesis reactions were performed in order to substitute the elevenlysine residues of carboxyl esterase (see FIG. 1) one at a time by aglutamine residue. In addition a twelveth reaction was carried out inwhich a mixture of eleven different oligonucleotides, each encoding adifferent lysine→ glutamine mutation, was included into the mutagenesisprotocol. The resulting mutant esterase from reaction 1-11 was producedin E. coli DHI (ATCC 33849) as described in EP-A-233656 (with thedeviation that vector pTZ18R instead of pUN121 was used) and tested forstability in the presence of (S)-naproxen (as described in Example 3).The mixture of mutants from reaction 12 was distributed into microtiterplates and tested for stability in the presence of naproxen using acolor assay based on β-naphthol and fast-blue to determine residualactivity of the mutant esterases. An automatic pipetting robot was usedto screen 20.000 candidate mutants. More stable mutant enzymesoriginating from the twelve different mutagenesis reaction were selectedand used for further characterization.

EXAMPLE 3 Stability of mutant carboxyl esterases.

Mutant carboxyl esterases constructed as described in Example 2 weretested for stability as follows: a solution of 9 ml containing 0.10 g(S)-naproxen was incubated at 40° C. for 15 minutes prior to theaddition of 1 ml containing 24 U carboxyl esterase. The composition ofthe final mixture was 10 g/l naproxen, 1 mM MOPS, 20 mM glycine, 2.4U/ml carboxyl esterase, pH 8.75. Immediately after addition of theenzyme solution and mixing, the first 50 μl sample was drawn (0 minutesample). At 0, 15, 30, 45, 60, 90, 120, 180 and 240 minutes incubation50 μl samples were drawn and immediately diluted to 5 ml in 100 mM MOPSbuffer pH 8.75 containing 0.2% BSA. Carboxyl esterase activity wasassayed in these samples as described hereinbelow according to`Analytical Methods`.

Several mutant enzymes with increased stability in the presence ofnaproxen were obtained, for instance mutants in which Lysine 34, Lysine81 or Lysine 217 were replaced by Glutamine.

The majority of the constructed mutants showed decreased stability ordecreased activity, as expected. However, the observation that out ofeleven possible lysines, mutation of each of three positions can giverise to increased stability while retaining the activity of the enzyme,indicates the possibilities of site-directed mutagenesis. Furthermore,in this Example only mutations to glutamine were constructed.Replacement of lysines by other residues could also give good or evenbetter results. In this respect, substitution of lysines by arginineswould be preferred for the following reasons:

1. Arginine has the same positive charge as lysine;

2. The arginine epsilon amino group is less susceptible to modificationby alkyl or carboxyl groups than that of lysine.

Also construction of better mutants by combination, based on the onesnow available is feasible.

The inactivation profiles, as determined by assaying enzyme activityafter increasing periods of incubation of the enzyme with 10 g/lnaproxen as described above, is shown for three Lysine → Glutaminemutants and the wild-type enzyme in Table 1.

                  TABLE 1                                                         ______________________________________                                        Rest Activity                                                                 Time (min.)                                                                           Wild type Lys 34 Glu                                                                              Lys 81 Glu                                                                            Lys 217 Glu                               ______________________________________                                         0      100       100       100     100                                       15      79        93        67      87                                        30      56        81        63      71                                        45      43        81        54      60                                        60      34        75        49      53                                        90      24        68        42      38                                        120     14        61        25      26                                        180      6        49        13      15                                        240      4        38         8       8                                        ______________________________________                                    

These results are also depicted in FIG. 2.

Analytical Methods

Carboxyl esterase is assayed in 0.1 M MOPS(3-[N-morpholino]propanesulfonic acid) pH 7.5 at 25° C. in the presenceof 0.3 mg (S)-naproxen methyl ester per ml, Tween 80 (TM), 1 mg/ml BSA(Bovine Serum Albumin). The HPLC system used is a reversed phase column(Novapak CN Radial Pak cartridge from Waters) eluted with acetonitrile:0.03 M phosphate (34:66) pH 3.2 at a flow of 5 ml/min. Retention timesfound were 6.9 min for the methyl ester and 4.6 min for naproxen. 1 Unit(U) is defined as the amount of enzyme that hydrolyses 1×10⁻⁶ mol(S)-naproxen methyl ester per minute under the conditions as specifiedbelow.

EXAMPLE 4 Carboxyl esterase preparation from Bacillus subtilis1-85/pNAPT-7 and Bacillus licheniformis T9.

Bacillus subtilis 1-85/pNAPT-7 (CBS 673.86) was grown as described inthe European patent application EP-A-233656. The enzyme was isolated asdescribed in Example 14 of that application. The ultrafiltrationconcentrate was lyophilized. The activity of the dried material wasapproximately 2400 U/g.

In another experiment pNAPT-7 was transformed into Bacilluslicheniformis T9 using a protocol as described in EP-A-253455. Thisstrain, which is protease negative, α-amylase negative and sporulationnegative, is advantageous for fermentation and recovery of carboxylesterase. The enzyme was obtained analogously as the esterase fromBacillus subtilis 1-85/pNAPT-7 and showed a similar activity. Theactivities are determined according to the "Analytical Methods" ofExample 3.

EXAMPLE 5 Modification of carboxyl esterase by formaldehyde.

Carboxyl esterase (originating from Bacillus subtilis 1-85/pNAPT-7 (CBS673.86)) solutions were prepared containing 40 mg/ml lyophilized enzyme,250 mM MOPS (3-[N-morpholino]propanesulfonic acid) pH 7.5 and increasingformaldehyde concentrations (0.01-10%). The concentrations of themodifying agent are given as v/v% of the reaction mixture. The solutionswere left to stand at 20° C. for one hour. Subsequently, part of thesample was used for direct enzyme activity determination and part of thesample was first incubated with 15 mg/ml (S)-naproxen at 40° C. for 1.5hours before enzyme activity determination. The results are given inTable 2.

By rest activity is meant the activity left after a certain chemicaltreatment, relative to the activity without chemical treatment. Theactivities are determined according to the "Analytical Methods" ofExample 3.

                  TABLE 2                                                         ______________________________________                                                                  Rest activity after                                           Rest activity after                                                                           formaldehyde treat-                                 Formaldehyde                                                                            formaldehyde treatment                                                                        ment and incubation                                 %         (in %)          with naproxen (in %)                                ______________________________________                                        0         100              2                                                  0.01      105              7                                                  0.025     99               7                                                  0.05      100              8                                                  0.1       95              20                                                  0.25      93              45                                                  0.5       87              60                                                  1.0       64              61                                                  2.5       47              45                                                  5.0       36              41                                                  0.0       11              16                                                  ______________________________________                                    

The results are also depicted in FIG. 3. It is shown that the untreatedenzyme is completely inactivated on incubation with naproxen for 1.5hours at 40° C. Formaldehyde treatment of the esterase gives rise to apartial loss of activity. However, the modified enzyme, treated withformaldehyde concentrations of 1% or higher, is completely stable onincubation with naproxen (15 mg/ml) for 1.5 hours at 40° C.

EXAMPLE 6 Modification of carboxyl esterase with formaldehyde.

Carboxyl esterase (originating from Bacillus subtilis 1-85/pNAPT-7 (CBS673.86)) solution containing 10 mg/ml lyophilized enzyme, 250 mM MOPS,pH 7.5 and 2% formaldehyde was stirred at 20° C. for one hour. Thesample was dialyzed against 100 mM MOPS pH 7.5. The esterase activitywas determined according to the "Analytical Methods" of Example 3; therest activity was 60%.

EXAMPLE 7 Modification of carboxyl esterase with glutaraldehyde.

Carboxyl esterase (originating from Bacillus subtilis 1-85/pNAPT-7 (CBS673.86)) solution containing 10 mg/ml lyophilized enzyme, 250 mM MOPS,pH 7.5 and 2% glutaraldehyde was stirred at 20° C. for one hour. Thesample was dialyzed against 100 mM MOPS pH 7.5. The esterase activitywas determined according to the "Analytical Methods" of Example 3; therest activity was 68%.

EXAMPLE 8 Modification of carboxyl esterase with glyoxal.

Carboxyl esterase (originating from Bacillus subtilis 1-85/pNAPT-7 (CBS673.86)) solution containing 20 mg/ml lyophilized enzyme, 250 mMcarbonate, pH 9.2 and 0.8% glyoxal was stirred at 20° C. for one hour.The sample was dialyzed against 250 mM carbonate pH 9.2. The esteraseactivity of the material was determined according to the "AnalyticalMethods" of Example 3; the rest activity was 45%.

EXAMPLE 9 Modification of carboxyl esterase with succinic anhydride.

Carboxyl esterase (originating from Bacillus subtilis 1-85/pNAPT-7 (CBS673.86)) solution containing 10 mg/ml lyophilized enzyme, 0.5 M MOPS, pH8.0 and 0.3% succinic anhydride was stirred at 20° C. for one hour. Theesterase activity was determined according to the "Analytical Methods"of Example 3; the rest activity was 62%.

EXAMPLE 10 Modification of carboxyl esterase with glutaric anhydride.

Carboxyl esterase (originating from Bacillus subtilis 1-85/pNAPT-7 (CBS673.86)) solution containing 10 mg/ml lyophilized enzyme, 0.5 M MOPS, pH8.0 and 0.3% glutaric anhydride was stirred at 20° C. for one hour. Theesterase activity was determined according to the "Analytical Methods"of Example 3; the rest activity was 72%.

EXAMPLE 11 (R,S)-naproxen methyl ester conversion with modified carboxylesterase.

300 mg of (R,S)-naproxen methyl ester was added to 10 ml 2% Tween 80(TM). The pH was adjusted to 9.0. Subsequently 5.5 U of modified enzyme,prepared as described in Examples 6-10, were added. The pH was kept at9.0 by titration with 2.5 M ammonium hydroxide. The reaction was carriedout at 40° C. The extent of conversion was followed in the time by HPLC.A conversion with unmodified enzyme was used as a reference. Theresults, depicted in FIG. 4, show that the modified enzymes reach a muchhigher conversion than the untreated enzyme.

EXAMPLE 12 (R,S)-diclofop ethyl ester conversion with glutaraldehydemodified carboxyl esterase.

750 mg (R,S)-diclofop ethyl ester was added to 25 ml 1% Tween 80 (TM).The pH was adjusted to 9.0. Subsequently 10 U of modified enzyme wereadded. The modified enzyme was obtained as described in Example 7 exceptthat only 0.15% of glutaraldehyde was added. The pH was kept at 9.0 bytitration with 0.1 M NaOH. The temperature was 20° C. The extent ofconversion was followed in the time by HPLC. A conversion withunmodified enzyme was used as a reference. The results, depicted in FIG.5, show that the modified enzyme reaches a much higher conversion thanthe untreated enzyme.

We claim:
 1. A modified carboxyl esterase obtained from a strain ofBacillus and capable of stereospecifically hydrolyzingS-naproxen;wherein said modified carboxyl esterase shows enhancedstereospecific hydrolysis of (R,S)-naproxen esters and enhancedstability for naproxen when contacted with 15 mg/ml of (S)-naproxen atabout pH 9 and at 40° C. for 1.5 hours as compared to the correspondingunmodified wild-type carboxyl esterase, wherein said wild-type carboxylesterase is encoded by a DNA at least 70% homologous to the DNA of FIG.1 or wherein said wild-type carboxyl esterase is at least 70% homologousto the Bacillus subtilis Thai I-8 carboxyl esterase; and wherein saidmodified carboxyl esterase differs from said wild-type esterase in atleast one positively charged basic amino acid residue present in saidwild-type carboxyl esterase, which is replaced by a neutral ornegatively charged amino acid residue.
 2. Modified carboxyl esteraseaccording to claim 1 wherein said basic amino acid residue is a lysine,arginine or histidine residue.
 3. Modified carboxyl esterase accordingto claim 2 wherein said modified carboxyl esterase differs at said basicamino acid residue corresponding to any of Lys 34, Lys 81 or Lys 217 ofFIG.
 1. 4. Modified carboxyl esterase according to claim 1 wherein saidbasic amino acid residue is replaced by Glutamine.
 5. Modified carboxylesterase according to claim 1 whereby the wild-type carboxyl esterase isidentical, or substantially identical to the carboxyl esterase obtainedfrom a Bacillus subtilis strain.
 6. A method to conduct stereospecifichydrolysis of an ester which method comprises contacting said ester withan amount of the modified carboxyl esterase of claim 1 effective toresult in said stereospecific hydrolysis.
 7. A method according to claim6 wherein said ester is an (R,S)-2-substituted propionic acid ester. 8.A method according to claim 7 wherein said ester is naproxen, ibuprofenor diclofop ester.
 9. A DNA molecule comprising a nucleotide sequenceencoding the modified carboxyl esterase of claim
 1. 10. An expressionsystem for a recombinant host cell, capable of expressing a DNA encodingthe modified carboxyl esterase of claim 1, which expression systemcomprises a nucleotide sequence encoding said carboxyl esterase operablylinked to control sequences for effecting its expression.
 11. Arecombinant host cell modified to contain the expression system of claim10.
 12. Modified carboxyl esterase according to claim 5 wherein saidmodified carboxyl esterase is obtained from the strain Bacillus subtilisThai I-8 (CBS 679.85).
 13. A method to produce a modified carboxylesterase obtained from a strain of Bacillus and capable ofstereospecifically hydrolyzing S-naproxen that is modified with respectto wild-type carboxyl esterase which shows enhanced stereo specifichydrolysis of (R,S)-naproxen esters and enhanced stability for naproxenwhen contacted with 15 mg/ml of (S)-naproxen at about and at 40 degreesC for 1.5 hours as compared to the corresponding unmodified wild-typecarboxyl esterase, which method comprisesmodifying the gene encodingsaid wild-type esterase to replace the codon for at least one positivelycharged basic amino acid residue with a codon for a neutral ornegatively charged amino acid residue to obtain a modified gene; andexpressing the resulting modified gene to produce said modified carboxylesterase.
 14. A modified carboxyl esterase prepared by the method ofclaim 13.